The long-term goal of the proposed studies is to determine the physiological role(s) of the regulatory light chains of myosin (RLC) in the regulation and/or modulation of skeletal muscle contraction. The central hypothesis to be tested is that Ca2+ and/or Mg2+ binding to the single Ca2+ binding site on the RLC plays a role in the regulation and/or modulation of contraction. In order to test this hypothesis, the following Specific Aims will be pursued: I. What are the Ca2+ and Mg2+ binding properties of the single Ca2+ binding site on the RLC in muscle? To fully understand how metal (Ca2+ and/or Mg2+) binding to RLC might affect contraction, the metal binding properties of RLC in muscle will be measured. II. Does Ca2+ binding to the RLC affect the rate of force development and/or relaxation and, if so, how? Several lines of evidence suggest that Ca2+ binding to the RLC Ca2+ binding site affects the rate of force development, by somehow altering cross-bridge kinetics. The proposed experiments will test this idea by determining whether Ca2+ binding influences the rate of force development and/or relaxation. If, as expected, metal binding to the RLC plays a role in contraction, then changing the metal binding properties of the RLC should change the metal dependency of any affected contractile process. A series of RLC Ca2+ binding site mutants (e.g., inactivated site, higher Ca2+ affinity/ specificity site, etc.) will be incorporated into skinned muscle fibers and tested for their effects on a) steady state force development, b) the Ca2+-dependence of force development, c) the rate of activation/ relaxation, and d) the Ca2+ dependence of the rate of force development. III: Does phosphorylation of RLC by MLCK affect the Ca2+ dependence and/or kinetics of force development and relaxation, and does RLC phosphorylation interact functionally with RLC metal binding? Our Preliminary Studies have shown that the RLC are mostly phosphorylated in isolated fibers, and that changing the level of phosphorylation has a much larger effect on the Ca2+ dependence of force development than had been previously appreciated. Our results also suggest that Ca2+ binding to the RLC is required to observe these effects of RLC phosphorylation. These results will be confirmed and extended to ask the following questions: 1) are the effects of phosphorylation on the Ca2+-dependence of force development accompanied by changes in the kinetics of force activation/relaxation; 2) are the metal binding properties of the RLC affected by phosphorylation and vice versa and 3) are the effects of phosphorylation direct, due to changes in RLC Ca2+ binding, or indirect, through cross-bridge effects on Tn Ca2+ affinity? The answers to these questions will determine the mechanism and the importance of MLCK phosphorylation of RLC in skeletal muscle contraction. In summary, our experiments will define in detail the potential role of the RLC in the regulation and/or modulation of skeletal muscle contraction.